Responses of the Fish Blennius Pholis to Fluctuating Salinities

Vol. 1, 101-107, 1979 MARINE ECOLOGY - PROGRESS SERIES Published September 30 Mar. Ecol. hog. Ser.

Responses of the Fish Blennius pholis to Fluctuating Salinities

J. Davenport and 0. Vahl*

N.E.R.C. Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5EH. United Kingdom

ABSTRACT: Blennius pholis(L.) were exposed to fluctuating salinity regimes of near tidal periodicity and their blood osmolality, oxygen consumption, heart rate and opercular beat monitored. Despite salinity fluctuations from 34-0-34 '/m S, the blood osmolality remained constant. Significant increases in oxygen consumption were observed at low salin~tylevels, but these may simply reflect changes in physical activity and the greater availability of oxygen at low salinities. Salinity effects upon heart rate/ opercular beat were weak or negligible.

INTRODUCTION imposed upon animals by the salinity'of their environment. A number of invertebrates can effectively coun- The blenny Blennius pholis (L.)is a common inhabi- teract salinity stress during periods of each tidal cycle tant of rocky shores around Anglesey, U.K., particu- in littoral and estuarine areas, and thereby avoid or larly on the west coast of the island. It lives in rocky reduce damage by low environmental salinity levels. pools beneath stones, buried in muddy gravel and in Such compensations have been considered by Kinne rock crevices from just above the low water springtide (1967, p. 526) under the headings of escape, reduction level to the upper shore. Although it is a non-grega- of contact, regulation and acclimation. In several cirri- rious fish (Gibson, 1967a, 1968), its defended home pede, bivalve and polychaete species, compensation ranges are small, and thus local abundances may be for salinity stress has been studied by Davenport quite high. Blennies are particularly numerous in nar- (1976a), Davenport (1977) and Shumway and Daven- row rock gullies running at right angles to the coast, port (1977). As suggested by Kinne (1971b, p. 905) and and these are often fed by freshwater streams or see- Spaargaren (1974), invertebrates which cannot reduce page. Consequently the fish are exposed to considera- contact with adverse salinities tend to exhibit internal ble salinity fluctuations ranging from 33 to 34 O/OO S at osmolarity changes which are damped by comparison high tide, to freshwater at low tide. The individuals with those of the medium, and thus allow the animals living highest on the shore may be exposed to freshwa- to survive brief exposures to salinity levels which ter influence for several hours. would kill them if sustained for periods longer than The information available on the effects of salinity those prevailing in their natural environment. Such upon marine organisms in terms of their tolerance and damping has been confirmed by Tucker (1970) for the physiology has been reviewed extensively in 'Marine prosobranch Scutus breviculus, and for echinoderms Ecology', Volume I (Kinne, 1971a) and has received and bivalves by Stickle and Ahokas (1974) and Shum- detailed attention notably from Krogh (1939), Potts and way (1977). Data on fluctuating salinity effects upon Parry (1964) and Kinne (1971b). However, almost all of organisms have been obtained with the aid of an appa- the studies conducted thus far have involved labora- ratus which can mimic environmental salinity fluctua- tory experiments at constant salinities. tions: the most convenient and accurate apparatus is Steady-state salinity experiments are open to criti- that described by Davenport et al. (1975). cism on several grounds. Primarily they tend to give a Most of the information available thus far has been false impression of the possible limits to distribution obtained in invertebrates, largely for osmoconformers rather than osmoregulators. In teleost fishes the situa- On leave from Institute of Biology and Geology, University tion is different. They have a low blood concentration of Tromso, Norway. maintained by an efficient osmoregulatory mechanism

O by Inter-Research 102 Mar. Ecol. Prog. Ser., 1, 101-107, 1979

(e.g. Gilles, 1975) which, in euryhaline species, is rometric experiments described below, an abrupt responsible for the extrusion of salts at high salinity regime as described already, and a sinusoidal regime levels and for taking up salts at low seawater concen- of 12 hour wavelength fluctuating between full seawa- trations. The blenny appeared to be suitable for a first ter and fresh water. investigation with the fluctuating salinity apparatus, since this teleost is relatively small and its behaviour causes it to remain at positions on the shore which Heart/Opercular Beat Frequency Measurements expose it to local tidal salinity fluctuations. In the study reported here, blennies were exposed to Heart and opercular beat frequencies were both fluctuating salinity regimes of near-tidal periodicity, measured simultaneously in individual fish by the During this exposure their plasma osmolarity, oxygen impedance pneumograph technique (Trueman, 1967). consumption, heart rate and opercular beat frequen- Each fish was anaesthetised with MS 222 and a silver- cies were monitored. wire electrode sewn beneath the skin overlying each operculum. The two electrodes were connected, via fine polythene sheated copper wire to a Scientific MATERIALS AND METHODS Instruments' pneumograph coupler, itself connected to a twin channel George Washington 400 MD/2 chart Collection and Maintenance recorder. Another pair of silver electrode's were inser- ted through the body wall (either side of the heart); Blennies Blenniuspholis (L.) were mainly collected they were anchored in place by sewing to the bases of from the intertidal zone of the rocky west coast of the pelvic fins. These electrodes were also connected Anglesey (U.K.)between Aberffraw and Rhosneigr. A to a pneumograph coupler and the twin channel recor- few individuals were obtained from the shore of the der. The fish was then allowed to recover for several Menai Strait beneath the northern end of Telford hours before use in experiments. To conserve chart Suspension Bridge. The fish were collected from pools, paper during long experiments the power supply of the crevices and muddy gravel beneath stones. Fish in chart recorder was controlled by a time clock which pools were caught by the use of the anaesthetic quinal- switched the recorder on for 2 min every hour. Results dine (Gibson 196713). The blennies ranged in wet were obtained for 3 fish (19.0-27.5 g) exposed for 24 h weight from 7.4-42.6 g. To obtain arrythmic fish, the to the abrupt salinity regime. blennies were held in the laboratory seawater system at 10 "C and under constant illumination (Gibson 1967a). They were fed flesh of Mytilusedulis regularly, Plasma Osmolality Determinations but starved for 2 days before use in respirometry experiments. All experiments were performed in constant Blood samples were obtained by sacrificing fish at light at 10 "C. appropriate intervals during the first 12 h of an abrupt salinity regime. The tail of each fish was cut off and blood allowed to drip into a 1.5 m1 Eppendorf plastic Salinity Regimes tube. After centrifuging the blood to remove cells, a 50 mm3 sample of plasma was pipetted into the test vessel The blennies were exposed to salinity fluctuations of Knauer Semi-micro osmometer and its osmolality produced by the apparatus described by Davenport et established. Enough blood could be obtained from a al. (1975). To determine the influence of these fluctua- single large fish for such a determination, but on tions upon heart/opercular beat frequencies and several occasions the blood of 2-3 smaller fish had to plasma osmolarity the fish were placed in 500 m1 be pooled. plastic boxes supplied with water at 250-300 m1 min-l. The contents of the boxes were aerated by diffuser Respirometry stones connected to the compressed air supply. In these experiments only one type of salinity regime was used, Oxygen consumption measurements were carried namely an abruptly fluctuating cycle in which the test out by the use of the apparatus shown in Figure 1. Air- fish received alternate 6 h exposures to full seawater saturated water was drawn by a peristaltic pump from (33.5 '/W S) and fresh water. No more gradual regimes a column supplied with water of fluctuating salinity by than this were employed because the monitored phy- the apparatus described by Davenport et al. (1975). It siological changes induced by the abrupt salinity fluctuations were either slight or negligible. Scientific Instrument Centre, 13 Derby Lane, Lverpool, Two types of salinity regime were used in the respi- U.K. Davenport and Vahl: Responses of Blennius pholis to fluctuating salinites

column Fig. 1. Experimental set-up for measurement of oxygen consumption in various salinity regimes was then delivered to a 250 m1 glass experimental (5) The fish was removed from the respirometer and chamber fitted with a screw top plastic-coated metal weighed. lid, and magnetically stirred. From the experimental To avoid potential inaccuracies caused by oxygen- chamber the water flowed past a Radiometer E5046 electrode response drifts, the electrode was regularly oxygen electrode, connected via a Radiometer PHM 7 1 moved from its position in the experimental-chamber Mk I1 pH meter to a Smiths Servoscribe chart recorder outflow and placed for a few minutes in the surround- adjusted to 100 mV. The water then flowed through a ing temperature-controlled bath. Since this bath is platinum conductivity cell, connected via a Carwyn continually aerated, and at the same temperature as Instruments' Salinity Monitor to the same chart recor- the experimental chamber and aeration column, this der as the oxygen electrode. Thus a nearly simultane- procedure allows regular reassessment of the chart ous trace of the salinity and oxygen tension of the deflection corresponding to full air saturation. When water leaving the experimental chamber was avail- the flow rate of water through the respirometer is able. known, the oxygen uptake by the fish can be calcu- To carry out an experiment the following experimen- lated from the difference in oxygen saturation between tal protocol was adhered to: inflowing and outflowing water from the experimental (1) Air-saturated full seawater (33.5 @/m S) was chamber: the oxygen uptake was calculated for every allowed to flow through the apparatus for several hour of each of the 24 h periods, taking into account hours, and measurements were made to confirm that that the oxygen concentration of 100 '/e air-saturated temperatures remained the same in the temperature water varies with salinity. controlled bath, aeration column and outflow. For each hourly interval of the salinity regimes the (2) The oxygen electrode was zeroed and adjusted so regression of the logarithm of the weight-specific oxy- that 100 O/O seawater air saturation was equivalent to gen uptake on the logarithm of wet weight was calcu- near full-scale deflection on the chart record (for lated. Thus a number of regressions were obtained, details of calibration procedure see Davenport, 1976b). each relating oxygen consumption at a particular (3) A fish was placed in the experimental chamber salinity to the size of the fish. These data were further and allowed to settle down. The flow rate through the treated by pooled regression analysis and analysis of experimental chamber was adjusted, if necessary, so variance (ANOVA). 7 fish were exposed to abruptly that the resting fish removed 10-15 '/a of the oxygen in fluctuating salinity regimes and 14 to sinusoidal the water passing over it. cycles. (4) When the fish had become quiet and had shown no systematic decrease in respiration rate for several hours, the programmer of the salinity apparatus was RESULTS set to deliver an abrupt or sinusoidal regime over the ensuing 24 h period. Heart/Opercular Beat Frequency

Carwyn Instruments, Pentraeth Road, Menai Bridge, Gwy- In the abrupt salinity regime the fish were exposed nedd, U.K. to alternate periods of full seawater strength (34 '/no) 104 Mar. Ecol. Prog. Ser., 1. 101-107, 1979

and fresh water (0 "/(W]. Each period lasted for 6 h, so that each fish was exposed to 12 h of full strength Txand 95 0L c. I. mosmol seawater and 12 h of fresh water. It appears that when rned~urn tested as a group the heart beat frequencies differed significantly between individual fish and between .- salinities. There is probably no interaction (0.10 > P > r-- 0.05); this indicates that the individuals are affected - equally by salinity. All three fish showed signs of slight bradycardia in fresh water. However, a significantly lower heart beat frequency could only be 400 demonstrated for one of the blennies (Tabie 1).

Table 1. Blennius pholis. Differences in heart beat frequencies in full salinity (34 Oh)and in fresh water. 12 observations in all cases. - not significant, +, ++ and ++ + significant at 2, 5 and 1 'ic,levels, respectively Fig. 2. Blennius pholis. Changes in the osmolarity of blood Fish Wet Sali- Heart beats t- Level of plasma during abrupt changes in salinity from full seawater No. weight nity - min-' value signifi- to fresh water c.i.: confidence interval (9) ("l*) X S cance

34 61.50 1.88 0 60.42 2.43 Respirometry 34 71.00 3.30 2 21.5 0 65.83 5 39 In an abrupt salinity regime, pooled regression ana- 34 72.25 27'5 0 2.99 4,280 +++ lysis of the total mass of data of oxygen consumption by 67.58 2.31 the blenny (Fig. 3) indicates significant differences between regression coefficients (slopes) and between Table 2. Blennius pholis. Opercular beat frequencies constants (intercepts). The regressions were therefore sorted into groups according to the part of the regime Fish Wet Opercular beats min-' Number of they were obtained from. It then appears that there observations No. weight - were no significant differences between coefficients (9) X S and constants, except in the second period at full 1 19.0 36 4 3 24 salinity (9-15 h after start of the experiment). There- 2 21.5 66 2 1 23 fore, all the regressions for the weight-specific oxygen 3 27.5 4 8 43 24 consumption on weight in fresh water may be pooled to give a common regression. Similarly, the regressions Whereas the heart beats with a frequency of about for full salinity may be pooled for the experimental 60-70 beats min-l (Table l), the opercular beat fre- periods 0-3 and 21-24 h after the start of the experi- quency seems slower (Table 2). ANOVA reveals that in ment. Between these two pooled regressions there is a none of the fish did the variations in opercular beat significant difference in coefficients, indicating that frequency follow the variations in heart beat fre- large and small fish react differently to fresh water. quency. Visual observations and traces from the chart The regressions for the second period at full salinity recorder showed, however, that on occasion the oper- (8-15 h after start of the experiment) cannot be pooled cular movements stopped completely. This explains because of the significant differences among them. the large standard deviations shown in Table 2. These Since this implies that the number of degrees of free- large variations might have masked a possible covaria- dom is not increased, the confidence limits (Fig. 3) are tion of heart and opercular beat frequencies. wider here than for those periods where the regressions could be pooled, a procedure which increases the number of degrees of freedom. Plasma Osmolarity In the sinusoidal regime (Fig. 4) the blennies were exposed to the same salinities four times throughout In an abrupt salinity regime Blennius pholisshowed the 24 h cycle. Two of these were experienced in a great constancy of blood osmoconcentration. Although decreasing part of the regime and two in an increasing the medium changed from full-strength seawater to part. The pairs of regressions for each salinity in the fresh water, there occurred no significant (0.50 > P > decreasing part of the regime were compared, as were 0.25) changes in the osmolarity of the plasma (Fig. 2). the pairs in the increasing part. It appears that a Davenport and Vahl: Responses of Blennius pholis to fluctuating salinites 105

Mean oxygen uptake and 95% conf. int of

c 80 '. 35 270 3 0 60 8 Ya, 25 .-_z m 50 -.-C a 20 c 40 Q, DZ 15 5 30 10

Fig. 3. Blennius pholis. Oxygen consumption of a 15.3 g individual during abrupt changes in salinity slightly significant difference (0.05 > P > 0.025) within tion throughout a 24 h sinusoidal salinity regime may these pairs could only be demonstrated in one case, be calculated for any size of blenny. This is illustrated viz. in decreasing salinity at 12.6 "/W. As this difference for a 15.3 g individual in Figure 4. This weight was occurs in one out of twelve regressions, the probability chosen since it was the mean weight of blennies used for this happening by chance is (1/12) X 100 = 8.3 */o. in a later study by Vahl and Davenport (1979). Therefore, it is probably justified to pool all pairs of regressions, thus obtaining 6 pairs in all. Each member of a pair obtained in the two decreasing parts of the CONCLUSIONS regime and in the two increasing parts of the regime, respectively. Pooled regression analysis showed that, The results obtained demonstrate that Blennius depending on whether the blenny was exposed to pholis is capable of maintaining a remarkable phy- increasing or decreasing salinity, there were signifi- siological stability when exposed to the most severe cant differences in the levels of oxygen consumption at salinity fluctuations likely to occur in its intertidal salinities of 29.0, 21.4 and 12.6 O/M. Based on these habitat. pooled regressions, the changes in oxygen consump- The constancy of blood osmoconcentration shown in

Mean oxygen uptake and 95% conf~dence lim~tsof 15.3 g animal Salinity 80 0 -

O' 2 6 1b lh 1; 16 1h ;0;2;4h Fig. 4. Blennius pholis. Oxygen consumption of a 15.3 g individual exposed to a sinusoidal salinity regime 106 Mar. Ecol Prog Ser

abrupt salinity regimes, where fish encounter fresh ated sinusoidally from 6.4 m1 O2 I-' at high salinity to water for 6 h pcriods was somewhat unexpected. 8.0 m1 0, I-' at the lowest salinity level. This change in House (1963) showed that blennies acclimated to 10";, concentration of about 25 "/(, is comparable in size to seawater for 48 h had blood sodium and chloride con- the change in respiration rates observed. However, it centrations some 70 'ltt less than fish acclimated to full should be pointed out that Holliday's hypothesis is seawatcr, and it appears that Blennius pholis cannot rather dubious, since oxygen tensions rather than oxy- maintain its blood concentration at the level held in gen concentrations control oxygen exchange. full seawater in media below the isosmotic concentra- Moreover, the oxygen consumption data presented are tion of about 40 ;'!#I s.w. However, House was also able assymetric in form; if the changes in uptake were to demonstrate that the blenny differed from most other solely influenced by oxygen concentration, it would be euryhaline teleosts e.g. Anquilla i.nguil1a (Keys 1933) expected that consurnptior, 7.vould vary sinusoidally and Salmo gairdnerii (Houston, 1957) in that it exhibits and uptake maxima would coincide with salinity a very rapid physiological response to salinity mlnima. These comments also apply generally to the changes, switching in less than 5 min from pumping results derived from the abrupt salinity regime experi- salts outwards across the gills in hyperosmotic media, ments. Here, however, is an additional problem in that to actively taking up salts from hypo-osmotic media (< the fish appeared to show a large increase in oxygen 40 SW.). This rapidity of response, which operates in uptake when they were returned to seawater after the both directions, means that the passive loss of salts first fresh water exposure, although the data were very caused by exposure to fresh water will be partially variable. This response could not be demonstrated offset by active salt uptake, so that the blood osmocon- after the second fresh water exposure and may reflect centration will decline relatively slowly. In the experi- an increase in physical activity of the fish. Overall it ments reported here, it is clear that a 6 h exposure to would appear, therefore, that blennies take up more fresh water is insufficient to produce a statistically oxygen when exposed to fluctuating salinity regimes significant decline in blood osmoconcentration - a than they do in full seawater, but the precise reasons situation which is analogous to the blood osmocon- for this remain obscure. The importance of the extra centration damping response previously shown for energetic cost of living in a fluctuating salinity envi- invertebrates. It also remains possible that control of ronment will be explored further in the following drinking rates, urine concentration and urine produc- paper dealing with apparent specific dynamic action tion rates may contribute to hemostasis, but no data to and feeding strategies (Vahl and Davenport, 1979). confirm this is available. Since the oxygen consumption changes observed The variations in oxygen consumption are somewhat were small it is not surprising that the effects of salinity difficult to interpret. In the sinusoidal salinity regime, fluctuations upon heart and opercular beat should be consumption is elevated at low and rising salinities but weak. In any case, changes in blood perfusion rate of drops back to 'routine' level (Fry, 1957) at high and the gills may alter oxygen uptake without disturbance falling salinities. It is possible that the elevation at low of the heart or opercular beat. salinity levels is associated with the cost of osmoregulation: the increased oxygen consumption as the salinity rises is less easy to explain, but may well reflect Acknowledgements. Thanks are due to Professor T. Schweder increased physical activity. Such an increase in physi- for help with the statistics and to The Norwegian Research cal activity is to be expected in a fish which is inactive Council for Science and the Humanities for a grant to one of us (0. V.). at low tide and feeds at high tide. Possibly, rising salinity acts as a cue for searching activity to be resumed, whereas falling salinities precipitate inactiv- LITERATURE CITED ity. It should be noted that the fluctuations in mean oxygen consumption are relatively small, the max- Davenport, J. (1976a) A comparative study of the behaviour imum increase in mean rate above routine being about of some balanomorph barnacles exposed to fluctuating 19 "/(l. seawater concentrations. J. mar. biol. Ass. U. K., 56, Kinne (1964) and Holliday (1971) have stressed that 889-907. Davenport, J. (1976b). A technique for the measurement of the oxygen content of water depends upon salinity and oxygen consumption in small aquatic organisms. Lab. Holliday has stated that, since the oxygen uptake of Pract , 25, 693-695. fish is, to a large extent, determined by the oxygen Davenport. J. (1977). A study of the effects of copper applied concentration, it is difficult to assess whether a change continuously and discontinuously to specimens of Mytilus in respiration rate is related to salinity or oxygen con- edulis (L.) exposed to steady and fluctuating salinity levels. J. mar biol. Ass. U. K., 57, 63-74. centration. In our experiments the oxygen content of Davenport, J., Gruffydd, L1. D. and Beaumont. A. R. (1975).An the air-saturated water supplied to the blennies fluctu- apparatus to supply water of fluctuat~ngsalinity and its Davenport and Vahl: Responses of Blennius pholis to fluctuating salinites

use in a study of the salinity tolerances of larvae of the Kinne, 0. (Ed.) (1971a). Marine Ecology, Vol. 1, Environmen- scallop Pecten maximus L. J. mar. biol. Ass. U. K., 55, tal Factors, Part 2, Wiley, London. 391409. Kinne, 0. (1971b). Animals: invertebrates. In 0. Kinne (Ed.), Fry, F. E. J. (1957). The aquatic respiration of fish. In M. E. Marine Ecology, Vol. 1, Environmental Factors, Part 2. Brown (Ed.), The Physiology of Fishes, Vol. 1. Academic Wiley, London. pp. 821-995. Press, New York. pp. 1-63. Krogh, A. (1939). Osmotic Regulation in Aquatic Animals, Gibson. R. N. (1967a). Studies on the movements of littoral Cambridge University Press, Cambridge. fish. J. Anim. Ecol., 36, 215-233. Potts, W. T. W. and Parry, G. (1964). Osmotic and Ionic Gibson, R. N. (1967b). The use of the anaesthetic quinaldine Regulation in Animals, Pergamon Press, London. in fish ecology. J. Anim. Ecol., 36, 295-301. Shumway, S. E. (1977). The effects of fluctuating salinity on Gibson, R. N. (1968). The agonistic behaviour of juvenile the osmotic pressure and Nat, Ca++ and Mg++ concen- Blennius pholis. Behaviour, 30, 192-217. trations in haernolymph of bivalves. Mar. Biol., 41, Gilles. R. (1975). Mechanisms of ion and osmoregulation. In 153-177. 0. Kinne (Ed.), Marine Ecology, Vol. 2, Physiological Shumway, S. E. and Davenport, J. (1977). Some aspects of the Mechanisms, Part 1. Wiley, London. pp. 259-347. physiology of Arenicola marina (Polychaeta) exposed to Holliday, F. G. T. (1971). Salinity: fishes. In 0. Kinne (Ed.), fluctuating salinities. J. mar. biol. Ass. U. K., 57, 907-924. Marine Ecology, Vol. 1, Environmental Factors, Part 3. Spaargaren, D. H. (1974). A study on the adaptation of marine Wiley, London. pp. 997-1083. organisms to changing salinities with special reference to House. C. R. (1963). Osmotic regulation in the brackish water the shore crab Carcinus maenas (L.). Comp. Biochem. teleost Blennius pholis. J. exp. Biol., 40, 87-104. Physiol., 47A, 449-512. Houston, A. H. (1957). Responses of juvenile chum, pink and Stickle, W. B. and Ahokas, R. (1974). The effects of tidal coho salmon to sharp seawater gradients. Can. J. Zool., 35, fluctuations of salinity on the perivisceral fluid composi- 37 1-383. tion of several echinoderms. Comp. Biochem. Physiol., Keys, A. B. (1933). The mechanism of adaptation to varying 47A, 469476. salinity in the common eel and the general problem of Trueman, E. R. (1967). Activity and heart rate of bivalve osmotic regulation. Proc. R. Soc. (Ser. B), 112, 184-199. molluscs in their natural habitat. Nature, Lond., 214, Kinne, 0. (1964). The effects of temperature and salinity on 832-833. marine and brackish water animals 11. Salinity and temp- Tucker, L. E. (1970). Effect of external salinity on Scutus erature - salinity combinations. Ocean. Mar. Biol. Ann. breviculus (Gastropoda, Prosobranchia). 1. Body weight Rev., 2, 281-339. and blood composition. Comp. Biochem. Physiol., 36, Kinne, 0. (1967). Physiology of estuarine organisms with 301-319. special reference to salinity and temperature: general Vahl, 0, and Davenport. J. (1979).Apparent specific dynamic aspects. In G. H. Lauff (Ed.), Estuaries. Am Ass. Adv. Sci., action of food in the fish Blennius pholis. Mar. Ecol. hog. Washington. pp. 525-540. Ser., 1, 109-1 13.

This paper was presented by Professor T. M. Fenc :hel; it was accepted for printing on August 8, 1979.